Immobilization of biomolecules

ABSTRACT

This invention relates to a biomolecule-bound substrate made of polymeric molecules, each of which contains a reacting group; and a plurality of biomolecules, each of which contains another reacting group. One of the two reacting groups is a substitute group and the other is a leaving group; and the biomolecules are covalently bonded to the solid support via a chemical ligation reaction between the two reacting groups.

BACKGROUND

[0001] Diagnostic assays and other chemical processes often requireattaching a molecule to a solid support. For example, a protein iscommonly attached to a solid support for an immune assay, and anoligonucleotide to a solid support for a hybridization-based assay. Theattachment can be achieved in a number of different ways, includingcovalent bonding and non-covalent interaction. Typically, covalentattachment is more robust. See, for example, Lamture et al. (1994)Oligonucleotide Research 22: 2121-2125; Beattie et al. (1995) Mol.Biotechnol. 4: 213-225; Joos et al. (1997) Anal. Biochem 247: 96-101;Rogers et al. (1999) Anal. Biochem. 266: 23-30; and Chrisey et al.(1996) Oligonucleotide Research 24: 3031-3039.

[0002] A number of protocols have been developed to covalently attach anoligonucleotide to a support surface. One example achieves this by,e.g., synthesizing an oligonucleotide directly on a support surfaceusing stepwise photolithographic reactions. For example, see U.S. Pat.Nos. 5,424,186; 5,510,270; and 5,744,305. Alternatively, a nucleic acid,such as a cloned cDNA, a PCR product, or a synthetic oligonucleotide,can be deposited onto a surface of a solid support, e.g., a microscopicglass slide, in the form of an array. Usually, the surface is modifiedin order to covalently attach a nucleic acid.

SUMMARY

[0003] In one aspect, this invention relates to a biomolecule-boundsubstrate that includes (1) a solid support made of polymeric molecules,each of which contains a reacting group; and (2) a plurality ofbiomolecules, each of which contains another reacting group. One of thetwo reacting groups is a leaving group and the other is a substitutegroup, and the biomolecules are covalently bonded to the solid supportvia a chemical ligation reaction between the two reacting groups. Inother words, after the chemical ligation reaction, the leaving groupdeparts from the biomolecules or the solid support, and the substitutegroup bridges the biomolecules and the solid support via a covalentbond. Each of the two just-described reacting groups refers to eitherits pre- or post-reaction state, depending on whether it participates inthe reaction. Indeed, in a biomolecule-bound substrate of thisinvention, not all reacting groups on the substrate participate in theligation reaction.

[0004] A leaving group is the group that departs from a molecule duringa chemical ligation reaction. It can be, for example, halogen. Asubstitute group can be either a nucleophilic group or an electrophilicgroup. A nucleophilic group is a chemical species having unshared pairelectrons (e.g., any Lewis base), and can be neutral or have a negativecharge. Examples of the nucleophilic group include an oxygen-containinggroup (e.g., hydroxyl, alkoxy, or acyloxy), a sulfur-containing group(e.g., mercapto, alkylthio, sulfonate, or phosphorothioate), anitrogen-containing group (e.g., amino, alkylamino, acylamino, nitro,azido, or isocyanato), and halogen. An electrophilic group is a chemicalspecies having a vacant orbital for electrons to occupy, and can beneutral or have a positive charge. An example of the electrophilic groupis an organometal.

[0005] A solid support used to practice this invention is made of atleast one type of polymeric molecules evenly distributed throughout thesolid support, each of which contains a leaving group or a substitutegroup. A solid support can be flexible and capable of being bent,folded, or otherwise manipulated without breakage. It can also be rigidand takes on a desirable configuration, such as film, sheet, tube, disc,or sphere. A porous solid support, such as gel, can also be used. Any ofthe just-described solid support can be used alone, or in combinationwith any other support (e.g., glass) well known in the art. Examples ofa solid support for use in this invention include, but are not limitedto, polyvinyl chloride resin (PVC), urea-formaldehyde resin, andacrylic. When a solid support is PVC resin, the chloride group in thePVC is a leaving group. A biomolecule containing a substitute group canreact with the chloride group, resulting in covalently bonding of thebiomolecule to the PVC resin. When a solid support is urea-formaldehyderesin, the amino group in urea-formaldehyde is a substitute group. Abiomolecule containing a leaving group can react with the amino group,resulting in covalently bonding of the biomolecule to theurea-formaldehyde resin.

[0006] A biomolecule to be attached to the just-described solid supportcan be a biopolymer (e.g., an oligonucleotide, a peptide, apolysaccharide, or a glycoprotein) or a biomonomer (e.g., a nucleoside,an amino acid, or a monosaccharide), any of which can be a naturallyoccurring molecule or a synthetic analog. The term “oligonucleotide”used herein refers a synthetic DNA, a synthetic RNA, a cDNA, an mRNA, ora peptide nucleic acid. The biomolecule contains a leaving group or asubstitute group, which, if not present naturally, must be introduced bychemical or biochemical methods well known in the art. A leaving groupor a substitute group can locate at any suitable position of thebiomolecule.

[0007] In another aspect, this invention relates to a method forpreparing the afore-described biomolecule-bound substrate. The methodincludes (1) providing a solid support made of polymeric molecules, eachof which contains a leaving group (or a substitute group); and aplurality of biomolecules, each of which contains a substitute group (ora leaving group); and (2) bonding the biomolecules to the solid supportvia a chemical ligation reaction between the leaving and substitutegroups to form a biomolecule-bound substrate.

[0008] One advantage of this invention is that the surface of a solidsupport need not be modified in order to covalently attach abiomolecule. Other advantages, features, and objects of the inventionwill be apparent from the description and from the claims.

[0009] The details of one or more embodiments of the invention are setforth in the description below.

DETAILED DESCRIPTION

[0010] A biomolecule-bound substrate of this invention can be preparedby covalently bonding a biomolecule containing a substitute group to asolid support containing a leaving group. For example, one can deposit aphosphorothioate-containing oligonucleotide onto PVC resin. Thephosphorothioate group, a substitute group, reacts with the chloridegroup, a leaving group, in the PVC to form an oligonucleotide-bound PVCresin. Shown below is a scheme that depicts this chemical ligationreaction.

[0011] A biomolecule-bound substrate of this invention also can beprepared by covalently bonding a biomolecule containing a leaving groupto a solid support containing a substitute group. For example, one candeposit an iodothymidine-containing oligonucleotide ontourea-formaldehyde resin. The amino group, a substitute group, in theurea-formaldehyde resin reacts with the iodo group, a leaving group, onthe oligonucleotide to produce oligonucleotide-bound urea-formaldehyderesin. Shown below is a scheme that depicts this chemical ligationreaction.

[0012] A biomolecule has a reacting group (i.e., a leaving group or asubstitute group), which can locate at any suitable position. Forexample, an oligonucleotide has a reacting group at its the 3′ or the 5′terminus, or at a non-terminal position. It can be immobilized onto asolid support and hybridize with its pair member. When the reactinggroup is at a non-terminal position, the oligonucleotide may be capableof forming a “hairpin” structure on the solid support to improvehybridization efficacy.

[0013] A biomolecule that contain a reacting group can be prepared usingany convenient methodology. For example, wherein the biomolecule is anoligonucleotide, a number of protocols exist for introducing anoligonucleotide with a reacting group, if not present naturally. Forinstance, an oligonucleotide can be chemically synthesized on a DNA/RNAsynthesizer using non-modified phosphoramidites, and a reacting groupcan be enzymatically added to one of the termini of the oligonucleotide.Alternatively, a modified phosphoramidite, with a reacting group, can beincorporated into a suitable position of an oligonucleotide using aDNA/RNA synthesizer. As another example, where the biomolecule is apeptide, it can be prepared chemically (e.g., on a peptide synthesizer)or biologically (e.g., expressed from a host cell). A functional groupsuch as carboxy, hydroxy, phenoxy, amino, guanidino, or mercapto ispresent in peptides, and can serve as a reacting group. If an additionalreacting group is needed, it can be introduced by, for example,incorporation of an amino acid analog.

[0014] Synthesis of a backbone-modified oligonucleotide, such as aphosphorothioate-containing oligonucleotide, is described in, forexample, Krieg et al. (1995) Nature 374: 546-549; Weiner et al. (1997)Proc. Natl. Acad. Sci. USA 94: 10833-10837; and Boggs et al. (1997)Antisense Nucleic Acid Drug Dev 7(5): 461-471. Synthesis of abase-modified oligonucleotide, as well as other backbone-modifiedoligonucleotides (e.g., containing phosphorodithioate oraminoalkylphosphotriester), is described in, for example, U.S. Pat. No.6,232,296.

[0015] A biomolecule can be bound to the solid support randomly, or inan order. The method of this invention can introduce a biomolecule, suchas an oligonucleotide, a peptide, a polysaccharide, a nucleoside, anamino acid, or a monosaccharide, on a solid support in the form of anarray, i.e., an orderly arrangement such as a matrix of rows andcolumns. An individual array can contain a number of unique attachedbiomolecules. The array may contain a plurality of addresses (eachaddress being a unique attached biomolecule), and one or more uniqueattached molecules. Each addressable site can be directly adjacent to atleast one other site, or can be separated from each other site, e.g., bya ridge, etch or surface lacking attached biomolecules. The array canhave a plurality of addresses on the solid support. The density of theaddresses is selected to provide for adequate resolution for detection,and can be at least 10, 10³, 10⁵, 10⁷ or 10⁹ addresses/cm², or no morethan 10, 10³ , 10⁵, 10⁷ or 10⁹ addresses/cm². The center to centerdistance between addresses can be 1 cm, 10 mm, 10 nm, 0.1 nm or less, orranges between. The longest diameter of each address can be 1 cm, 10 mm,10 nm, or ranges between. Each address can contain 10 mg, 100 ng, 100pg, 0.1 pg or less of the biomolecule, or ranges between. Alternatively,each address contains 100, 10⁴, 10⁶, 10⁸ or more biomolecules, or rangesbetween. The addresses can be distributed, on the substrate in onedimension, in two dimensions, or in three dimensions.

[0016] A biomolecule-bound substrate of this invention can be used invariety applications, where such applications are generally analyticalin which the presence of a particular analyte in a given sample isdetected at least qualitatively, if not quantitatively. Morespecifically, the sample suspected of containing the analyte of interestis contacted with the biomolecules on the solid support under conditionssufficient for the analyte to interact with its respective pair memberthat is present on the solid support. If the analyte of interest ispresent in the sample, it can form a complex with its pair member. Thepresence of the complex can be detected by, e.g., use of a signalproduction system such as an isotopic or fluorescent label present onthe analyte.

[0017] An example of a biomolecule-bound substrate is an oligonucleotidearray, in which a hybridization assay can be employed. The hybridizationassay can be a gene discovery assay, a differential gene expressionanalysis assay, a sequencing assay, or an analysis of genomicpolymorphism. For example, an oligonucleotide array can be used toproduce gene expression profiles after polymerase mediated primerextension reactions. See, e.g., U.S. Pat. No. 5,262,311; Liang, P &Pardee, A. B. (1992) Science 257; 967-971; Liang, P & Pardee, A. B. eds.(1997) Methods in Molecular Biology: Differential Display Methods andProtocols, Vol 85.). Such an array can be used, for example, to identifygenes associated with diseases or screen compounds for drug discovery ina high throughput format. In particular, a high-density array has beenproven to monitor gene expression, map genomic library clones, andresequence genes to screen for mutations and polymorphisms. For example,see, Ramsay (1998) Nature Biotechnology 16: 40-44; Bains and Smith(1988) J. Theor. Biol. 135:303-307; Drmanac et al. (1989) Genomics4:114-128; and Shena et al. (1995) Science 270:467-470.

[0018] The specific examples below are to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. Without further elaboration, it is believed that oneskilled in the art can, based on the description herein, utilize thepresent invention to its fullest extent. All publications, includingpatents, cited herein are hereby incorporated by reference in theirentirety.

EXAMPLE 1 Preparation of An Oligonucleotide-Bound Plastic Substrate.

[0019] A 10 μM 3′-phosphorothioate and 5′-biotin modifiedoligonucleotide solution was prepared. 0.5 μL of the solution wasspotted onto a plastic substrate made of PVC resin. The plastic chip wasincubated at 32° C. overnight, washed with deionized distilled water(DDW), reacted with streptavidin-alkline phosphatase, and treated with abuffer containing nitroblue tetrazolium(NBT)/5-bromo-4-chloro-3-indolylphosphate (BCIP). The results indicatethat oligonucleotide was covalently bound to the plastic substrate.

Example 2 Preparation Of An Oligonucleotide-Bound Substrate.

[0020] A 10 μM 3′-amino and 5′-biotin modified oligonucleotide solutionwas prepared. 0.5 μL of the solution was spotted onto a plastic chipmade of PVC resin. The plastic substrate was incubated at 32° C.overnight, washed with DDW, reacted with streptavidin-alklinephosphatase, and treated with a buffer containing NBT/BCIP. The resultsindicate that oligonucleotide was covalently bound to the plasticsubstrate.

EXAMPLE 3 Hybridization Of A Bound Oligonucleotide On A PlasticSubstrate.

[0021] A 10 μM 3′-phosphorothioate modified oligonucleotide solution wasprepared. 0.5 μL of the solution was spotted onto a plastic chip made ofPVC resin. The plastic substrate was incubated at 32° C. overnight, andwashed with DDW. A biotin-labeled PCR product, containing a sequencecomplementary to the 3′-phosphorothioate modified oligonucleotide, wasapplied to the plastic substrate. After incubation, the chip was washed,reacted with streptavidin-alkline phosphatase, and treated with adetection buffer containing NBT/BCIP. The results showed thathybridization of the PCR product to the oligonucleotide on the substratewas efficient.

OTHER EMBODIMENTS

[0022] All of the features disclosed in this specification may be usedin any combination. Each feature disclosed in this specification may bereplace by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

[0023] From the above description, one skilled in the art can easilyascertain the essential characteristics of the present invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions. Accordingly, other embodiments are also within the scopeof the following claims.

What is claimed is:
 1. A biomolecule-bound substrate comprising: a solidsupport made of polymeric molecules, each of which contains a firstreacting group; and a plurality of biomolecules, each of which containsa second reacting group; wherein one of the two reacting groups is asubstitute group and the other is a leaving group; and the biomoleculesare covalently bonded to the solid support via a chemical ligationreaction between the two reacting groups.
 2. The biomolecule-boundsubstrate of claim 1, wherein the first reacting group is a leavinggroup and the second reacting group is a nucleophilic group.
 3. Thebiomolecule-bound substrate of claim 2, wherein the first reacting groupis halogen, amino, or mercapto.
 4. The biomolecule-bound substrate ofclaim 3, wherein the solid support is polyvinyl chloride and the firstreacting group is chloride.
 5. The biomolecule-bound substrate of claim2, wherein the second reacting group is phosphorothioate or an amino. 6.The biomolecule-bound substrate of claim 5, wherein the first reactinggroup is halogen, amino, or mercapto.
 7. The biomolecule-bound substrateof claim 6, wherein the solid support is polyvinyl chloride and thefirst reacting group is chloride.
 8. The biomolecule-bound substrate ofclaim 2, wherein the biomolecule is a nucleotide or an oligonucleotide.9. The biomolecule-bound substrate of claim 8, wherein the secondreacting group is phosphorothioate or an amino.
 10. Thebiomolecule-bound substrate of claim 9, wherein the first reacting groupis halogen, amino, or mercapto.
 11. The biomolecule-bound substrate ofclaim 10, wherein the solid support is polyvinyl chloride and the firstreacting group is chloride.
 12. The biomolecule-bound substrate of claim2, wherein the biomolecule is an amino acid or a peptide.
 13. Thebiomolecule-bound substrate of claim 1, wherein the first reacting groupis a leaving group and the second reacting group is an electrophilicgroup.
 14. The biomolecule-bound substrate of claim 1, wherein the firstreacting group is a nucleophilic group and the second reacting group isa leaving group.
 15. The biomolecule-bound substrate of claim 14,wherein the first reacting group is halogen, amino, or mercapto.
 16. Thebiomolecule-bound substrate of claim 15, wherein the solid support isurea-formaldehyde resin and the first reacting group is amino.
 17. Thebiomolecule-bound substrate of claim 14, wherein the second reactinggroup is halogen.
 18. The biomolecule-bound substrate of claim 17,wherein the first reacting group is halogen, amino, or mercapto.
 19. Thebiomolecule-bound substrate of claim 18, wherein the solid support isurea-formaldehyde resin and the first reacting group is amino.
 20. Thebiomolecule-bound substrate of claim 14, wherein the biomolecule is anucleotide or an oligonucleotide.
 21. The biomolecule-bound substrate ofclaim 20, wherein the second reacting group is halogen.
 22. Thebiomolecule-bound substrate of claim 21, wherein the first reactinggroup is halogen, amino, or mercapto.
 23. The biomolecule-boundsubstrate of claim 22, wherein the solid support is urea-formaldehyderesin and the first reacting group is amino.
 24. The biomolecule-boundsubstrate of claim 14, wherein the biomolecule is an amino acid or apeptide.
 25. The biomolecule-bound substrate of claim 1, wherein thefirst reacting group is an electrophilic group and the second reactinggroup is a leaving group
 26. A method for preparing a biomolecule-boundsubstrate, comprising: providing a solid support made of polymericmolecules, each of which contains a first reacting group; and aplurality of biomolecules, each of which contains a second reactinggroup; wherein one of the two reacting groups is a substitute group andthe other is a leaving group; and bonding the biomolecules to the solidsupport via a chemical ligation reaction between the two reacting groupsto form the biomolecule-bound substrate.
 27. The method of claim 26,wherein the first reacting group is a leaving group and the secondreacting group is a nucleophilic group.
 28. The method of claim 27,wherein the solid support is polyvinyl chloride and the first reactinggroup is chloride.
 29. The method of claim 27, wherein the secondreacting group is phosphorothioate or an amino.
 30. The method of claim29, wherein the solid support is polyvinyl chloride and the firstreacting group is chloride.
 31. The method of claim 27, wherein thebiomolecule is a nucleotide or an oligonucleotide.
 32. The method ofclaim 27, wherein the biomolecule is an amino acid or a peptide.
 33. Themethod of claim 26, wherein the first reacting group is a leaving groupand the second reacting group is an electrophilic group.
 34. The methodof claim 26, wherein the first reacting group is a nucleophilic groupand the second reacting group is a leaving group.
 35. The method ofclaim 34, wherein the biomolecule is a nucleotide or an oligonucleotide.36. The method of claim 34, wherein the biomolecule is an amino acid ora peptide.
 37. The method of claim 26, wherein the first reacting groupis an electrophilic group and the second reacting group is a leavinggroup.